Since “virus-like” particles were first discovered in adenovirus (AdV) preparations, adeno-associated virus (AAV) has been characterized and developed in the last 50 years as a potent viral vector to deliver genes in vitro in cultured cells and in vivo in animal models. AAV is a small, “naked” virus containing a single-stranded DNA genome of approximately 4.7 kb, consisting of two inverted terminal repeats (ITRs) that are capable of forming T-shape secondary structure and acting as origins of genome replication, one rep region that encodes four overlapping replication proteins, Rep78, Rep68, Rep52, and Rep40, and one cap region that encodes three structural proteins, VP1, VP2, and VP3. Naturally isolated serotypes 1-9 of the AAV viruses share the genomic structure although these serotypes may display different tissue tropism. Numerous investigations have revealed their attractive features including nonpathogenicity, efficient transduction and stable expression, thus laying foundations for recombinant AAV vectors for use as one of the most successful gene delivery vehicles.
Production of recombinant AAV vectors (rAAV) is traditionally achieved by transfection of human-derived HEK293 cells with a rAAV viral vector and a packaging construct in the presence of auxiliary viruses such as adenoviruses (AdV) that provide the helper function. After identification of AdV regions required for AAV vector packaging, a helper virus-free method was established using a constructed helper plasmid acting as auxiliary viruses. Therefore, a helper-free system is a triple transfection protocol consisting of three plasmids, which system is widely used in research and drug development. In addition, development of baculovirus expression vectors provides another method to produce rAAV viruses in insect sf9 cells. These different technologies are shown to be able to produce sufficient quantities of rAAV viruses for use in laboratories and clinical trials.
For purification of all of the different AAV serotypes, the viral particles are first released from the packaging cells using 3-4 freeze/thaw cycles, a conventional extraction method. In most purification protocols established to date, cesium chloride-(CsCl) and iodixanol-based density gradient ultracentrifugation is a central step. Although the gradient purification is useful in laboratory settings, it has raised concerns for clinical applications. Such concerns, in general, relate to (1) significant reduction of infectivity, (2) gradient-associated health risks, (3) gradient-associated toxicity, and (4) cumbersome procedures.
More recently, affinity and ion exchange chromatography have been tested for AAV particle purification. Such technologies, however, are associated with high cost and low scalability, or suffer from difficulties in separating viral particles from empty particles. Either affinity or ion exchange chromatography alone is unable to produce high purity of AAV viruses.